Pancreatic ductal adenocarcinoma, synonymous to pancreatic cancer, is the 4th leading cause of cancer deaths. The silent killer is characterized by its metastatic behavior before the primary tumor can be detected, resulting in a five-year survival rate of only 4%, underscoring the need for new alternative therapies. Listeria monocytogenes-based cancer therapy could be such an alternative therapy. Our laboratory discovered that Listeria exhibits novel pathways that are particularly useful against metastatic cancer. We found that Listeria infects metastases and primary tumors, and kills tumor cells through high levels of reactive oxygen species (ROS), and that myeloid-derived suppressor cells (MDSC) deliver Listeria selectively to the tumor site(s) through chemo/cytokines produced by the tumor cells, and by protecting them, after infection, from immune clearance through their immune suppressive character. Based on these results we now use Listeria as a platform for the delivery of anticancer agents to the tumor microenvironment (TME). One novel application is the delivery of radioisotopes, which emit cytocidal radiation such as beta-particles coupled to Listeria, selectively into the tumor cells. This leads to the synergistic destruction of tumor cell by ionizing radiation, which killed cells through beta-particles, and through ROS generation by Listeria. We were the first to demonstrate that in a highly aggressive model of pancreatic cancer (Panc-02), therapeutic treatment with Listeria reduced the number of metastases by 50%, and when coupled to radioisotope 188-Rhenium (188Re) by 90%. This correlated with a selective accumulation of radioactivity in the metastases with practically no side effects. This work was published in PNAS last year and its potential for human clinical trials was extensively discussed in a PNAS commentary, as well as in Science, Nature and lay magazines like The Economist and Forbes. Most recently, we explored a completely new method to RL generation using 32Phosphorus (32P), by incorporating 32P into the cell wall of Listeria (Listeria-32P) during culturing of Listeria. The Listeria-32P proved to be not only more effective against pancreatic cancer than Listeria-188Re (most likely as a result of the 32P longer half-life of 14 days vs 188Re of 17 hrs), but also much easier to generate. This novel approach avoids the need for antibodies and is much cheaper and faster than the generation of Listeria-188Re. Treatment with Listeria-32P completely eliminated the metastatic cancer in 80% of the mice. Most importantly, the incorporation of 32P delivered through Listeria into normal tissues including bone marrow (BM) was hardly detectable, in contrast to 32P alone, which strongly incorporated into the BM. Also, side effects of Listeria-32P were hardly detectable. In this grant application, our main goal is to explore the efficacy and safety of Listeria-32P for treatment of pancreatic cancer through evaluation in a humanized mouse model of pancreatic ductal adenocarcinoma (KPC mice, which conditionally express endogenous Kras-G12D and p53-R172H mutant alleles). The specific aims are as follows: (1) Evaluate function, stability, biodistribution, and safety of Listeria- 32P in KPC mice, and (2) Evaluate the effect of Listeria-32P on efficacy and survival in KPC mice.